Acta Microbiologica 27. (1980)
4. szám - Bánfalvi, G.–Ohlbaum, A.–Csuzi, S.–Antoni, F.: Purification and Characterization of a DNA-Dependent ATPase from Bacillus cereus
BÁNFALVI et al. HCl pH 7.5, 1 шм MgCl2, 2 шм 2-mercaptoethanol 30% v/v glycerol. SSC buffer: 150 mM NaCl, 15 mM sodium citrate pH 7.0. 3H—DNA was prepared from B. cereus 130 thy~ as described earlier [17]. Unlabelled DNA from chicken erythrocytes and yeast RNA were the products of Reanal (Budapest) PM 2 DNA was obtained from Dr. P. Medveczky (Institute of Microbiology, Semmelweis University Medical School), T7 DNA was obtained from Dr. A. Fekete (Institute of Biophysics, Semmelweis University Medical School). Heat dénaturation of DNA was carried out at 100 °C for 10 min in SSC buffer followed by rapid chilling in ice. DNA was sonicated by means of a Sonic 300 dismembrator (Artek) for 1 min in ice bath. UV light irradiated DNA was obtained by irradiation of native DNA by 100 000 erg/mm2 using a Germicid F lamp (Tungsram). Single stranded DNA from chicken erythrocytes was linked to CNBr-Sepharose 4B by the method of Arndt — Jovin et al. [18] as modified by Young [10]. y-32P-ATP (200 Ci per mole) was prepared according to Post and Sen [19]. Unlabelled ATP analogues, adenosine 5’-(/?, y-methylene)-diphosphate and adenosine 5’-(/?, y-imino)diphosphate were from Boehringer. Protein ivas determined by the method of Lowry et al. [20]; crystalline bovine serum albumin (Serva) was used as standard. IgG prepared against DNA helicase II from E. coli (50 mg per ml) was the gift of Dr. M. Abdel-Monem. Conditions for nuclease treatment of DNA. Incubation with pancreatic endonuclease (DNase I) in 0.3 ml contained 3 //moles of Tris-HCl buffer pH 7.5,1.5 nmole of MgCl2, 48 nmoles of 3H-labelled B. cereus DNA and 3 units of pancreatic deoxyribonuclease (Serva). The mixture was incubated at 35 °C for 30 min. The reaction was terminated by heating at 60 °C for 10 min. Acid soluble DNA was removed by dialysis against 200 volumes of SSC buffer. The dialysed material was used as DNase I treated DNA. The control sample was treated similarly except that nuclease was inactivated before addition to the incubation mixture. Sj nuclease was used to remove single stranded regions of DNA. The reaction mixture (0.3 ml) contained 10 //moles of sodium acetate pH 4.7, 50 //moles of NaCl, 0.3 //mole of ZnS04, 48 nmoles of 3H-labelled DNA and 20 units of Sx nuclease (Sigma). The mixture was incubated at 35 °C for 10 min. The reaction was terminated by heat inactivation and dialysed against SSC buffer as described above. Nuclease assay was carried out as described [5]. The reaction mixture contained in a volume of 0.15 ml, 10 nmoles of nucleotide equivalent 3H-DNA (7 X Ю3 cpm per nmole), 30 nmoles of ATP, 6 //moles of MgCl2, 7.5 //moles of Tris-HCl buffer pH 8.0 and enzyme. A control assay without ATP was always made. DNA-dependent ATPase assay was carried out by the measurement of 32Pj released from y—'32P-ATP. The reaction mixture (0.15 ml) contained 10 nmoles of DNA, 0.5 nmole of y—'32P-ATP (1-4 X 105 cpm per nmole), 150 nmoles of unlabelled ATP, 150 nmoles of MgCl2, 1.5 jumole of Tris-HCl buffer pH 8.0 and enzyme. After incubation at 35 °C for 20 min, the reaction was terminated by addition of 0.2 ml Norit A (Serva) suspension (4% in 0.1 M HC1) and 0.05 ml methanol. After shaking for one minute and standing in ice for 10 min, the charcoal was removed by centrifugation at 4000 g and the 32Pj remaining in the supernatant was measured in an aqueous solution by means of the Cerenkov effect. Parallel assay without DNA was performed. One unit means the amount of enzyme which degrades 1 nmole of ATP to ADP and Pj in 20 min under the above mentioned conditions. DNA duplex unwinding assay. The reaction mixture (0.3 ml) contained 10 nmoles 3H-DNA, 10 //moles Tris-HCl buffer pH 8.8, 1 //mole CaCl2, 30 nmoles ATP, 50 //g bovine serum albumin, 6-30 units of purified DNA-dependent ATPase (Fraction VI) and 2.5 mU micrococcal nuclease (Boehringer). The control reaction did not contain nuclease, ATPase or either of the enzymes. The reaction was incubated at 35 °C for 20 min and then terminated by the addition of 0.2 ml 2 м perchloric acid and 0.1 ml 5 mg per ml bovine serum albumin solution. Undigested DNA was precipitated and after standing at 0 °C for 10 min the sample was centrifuged at 4000 g for 10 min; 0.2 ml aliquots from the supernatant were pipetted into counting vials and radioactivity was measured. Results Purification of DNA-dependent ATPase. The procedure was similar to that used for purification of ATP-dependent DNase [5]. B. cereus NRRL B-569 was grown on Casamine medium [5]. The log phase cells (5xl08 cells Acta Microbiologica Academiae Scientiarum Hungaricae 27, 1980